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Abstract

Localization based super-resolution microscopy techniques require precise drift correction methods because the achieved spatial resolution is close to both the mechanical and optical performance limits of modern light microscopes. Multi-color imaging methods require corrections in addition to those dealing with drift due to the static, but spatially-dependent, chromatic offset between images. We present computer simulations to quantify this effect, which is primarily caused by the high-NA objectives used in super-resolution microscopy. Although the chromatic offset in well corrected systems is only a fraction of an optical wavelength in magnitude (<50 nm) and thus negligible in traditional diffraction limited imaging, we show that object colocalization by multi-color super-resolution methods is impossible without appropriate image correction. The simulated data are in excellent agreement with experiments using fluorescent beads excited and localized at multiple wavelengths. Finally we present a rigorous and practical calibration protocol to correct for chromatic optical offset, and demonstrate its efficacy for the imaging of transferrin receptor protein colocalization in HeLa cells using two-color direct stochastic optical reconstruction microscopy (dSTORM).

Figures (6)

Calculated optical offset as a function of decentration of a point-like source at three different excitation wavelength pairs and under three different optical conditions: microscope objective only (A), tilted dichroic mirror (B) and wedged emission filter (C).

Schematic view of the optical setup. Laser beams were expanded by lenses L1 (Thorlabs, AC127-025-A) and L2 (Thorlabs, AC254-150-A) and focused into the back focal plane of the microscope objective (O) by means of lens L3 (Thorlabs, AC508-250-A). Dotted and dashed lines depict the conjugate planes of the system. The image generated by the objective and tube lens (TL) was imaged into a CCD camera via a 1 × telescope formed by lenses L4 and L5 (Thorlabs, AC254-100-A). Multi-edge excitation (F1) and emission filters (F2) and a dichroic mirror (D) were applied to spectrally separate the excitation and emission light. The illumination condition was set via mirror M2 placed into the front focal plane of the focusing lens L3.

Localization of a single multicolored bead from every sub-region using 488 nm (blue), 561 nm (green) and 640 nm (red) excitation before (a) and after (b) optical offset correction. Each tile corresponds to an area of 240 x 240 nm2. Adjacent tiles are separated by 10 μm in the image plane, to provide representative samples across the entire FOV of 41 μm × 41 μm. Scale bars: 50nm.